Biocompatible Palladium Nanoparticles Prepared Using Vancomycin for Colorimetric Detection of Hydroquinone

Hydroquinone poses a major threat to human health and is refractory to degradation, so it is important to establish a convenient detection method. In this paper, we present a novel colorimetric method for the detection of hydroquinone based on a peroxidase-like Pd nanozyme. The vancomycin-stabilized palladium nanoparticles (Van-Pdn NPs, n = 0.5, 1, 2) were prepared using vancomycin as a biological template. The successful synthesis of Van-Pdn NPs (n = 0.5, 1, 2) was demonstrated by UV-vis spectrophotometry, transmission electron microscopy, and X-ray diffraction. The sizes of Pd nanoparticles inside Van-Pd0.5 NPs, Van-Pd1 NPs, and Van-Pd2 NPs were 2.6 ± 0.5 nm, 2.9 ± 0.6 nm, and 4.3 ± 0.5 nm, respectively. Furthermore, Van-Pd2 NPs exhibited excellent biocompatibility based on the MTT assay. More importantly, Van-Pd2 NPs had good peroxidase-like activity. A reliable hydroquinone detection method was established based on the peroxidase-like activity of Van-Pd2 NPs, and the detection limit was as low as 0.323 μM. Therefore, vancomycin improved the peroxidase-like activity and biocompatibility of Van-Pd2 NPs. Van-Pd2 NPs have good application prospects in the colorimetric detection of hydroquinone.


Introduction
Hydroquinone (HQ) is easy to react with peroxide free radicals and is commonly used in developers, hair dyes, pharmaceutical raw materials, and oxygen scavengers. However, HQ can inhibit the central nervous system and damage liver and kidney function, and it has been included in the list of three types of carcinogens. Therefore, it is necessary to establish an efficient and reliable HQ detection platform.
At present, there are many detection and analysis methods, such as colorimetric analysis [1,2], electrochemical analysis [3], and high-performance liquid chromatography [4]. Among them, the colorimetric analysis method has the advantages of easy operation, low cost, and good visibility. Therefore, colorimetry has become one of the main ways. In most colorimetric analyses of HQ, the addition of natural enzymes is required to catalyze the reaction. However, natural enzymes have disadvantages such as high cost and easy inactivation. Since the discovery of Fe 3 O 4 nanozymes [5,6], a variety of nanomaterials have been found to have enzyme-like properties. Nanozymes have good stability, high efficiency, and easy acquisition, so they are widely used in colorimetric detection. Many nanomaterials, such as MnO 2 [7], UiO-67-Cu 2+ [8], Co 3 O 4 nanoplates [9], and PPy NPs [10], have shown enzyme-like catalytic ability and have been used in colorimetric detection.

Enzyme-like Activity Characterization of Van-Pd n NPs
To determine peroxidase-like activity, 200 µL of Van-Pd n NPs (C Pd = 0.9 mM) and 300 µL of a 0.2 M pH = 4 acetic acid-sodium acetate buffer solution were mixed and incubated in a 2 mL PE tube. 1000 µL of a 0.2 M acetic acid-sodium acetate buffer solution containing 0.6 mM TMB and 100 µL of 0.03 M H 2 O 2 were mixed and incubated in a constant temperature mixer at 25 • C at 600 rpm for 2 min. Finally, UV-vis spectrophotometry was used to determine absorbance.
To explore the optimal reaction temperature of Van-Pd n NPs, 200 µL of Van-Pd n NPs (C Pd = 0.9 mM) and 300 µL of acetic acid-sodium acetate buffer solution at different pHs were added to a 2 mL PE tube, and 1000 µL of 0.2 M acetic acid-sodium acetate buffer solution at different pHs containing 0.6 mM TMB were mixed. The samples were incubated in a constant-temperature mixer at 25 • C at 600 rpm for 5 min. Finally, UV-vis spectrophotometry was used to determine the absorbance of the sample at 652 nm. The pH range was 1-12. The temperature range was 5-65 • C.
The catalytic kinetics of the system were studied using the following experimental approach or methodology: 200 µL of Van-Pd n NPs (C Pd = 0.9 mM) were added to a 2 mL PE tube. The absorbance of the sample over time at 652 nm was then tested using a UV-vis spectrophotometer. The amount of buffer solution was 1200-300 µL at 100 µL intervals; The amount of buffer solution containing TMB was 100-1000 µL at 100 µL intervals. The total amount of liquid was 1500 µL. The formula 1 was used to study the affinity for substrate and the maximum rate of the catalytic reaction during the catalytic process: Symbol description: K m -Michael's constant; V max -maximum reaction rate; [S]-substrate concentration.
Furthermore, the peroxidase-like mechanism was tested using terephthalic acid (TA) as a fluorescent probe with the following steps. First, four experimental groups were established: TA, TA + Van-Pd n NPs, TA + H 2 O 2 and TA + H 2 O 2 + Van-Pd n NPs. As for TA + H 2 O 2 + Van-Pd n NPs, 1000 µL of TA (C TA = 0.5 mM) was added into a 2 mL PE tube, and subsequently, 200 µL of an acetic acid-sodium acetate buffer solution with a pH of 4 was introduced into the same PE tube. The purpose of adding the buffer solution is to maintain a stable pH environment within the tube. This ensures that the conditions remain optimal for any subsequent steps or reactions that may take place. The fluorescence spectra of the final solution were measured using a fluorescence spectrometer to analyze the emission of light from the samples. The other control groups underwent the same experimental procedure as the samples being analyzed.

Determination of HQ Concentration
An amount of 50 µL of Van-Pd n NPs (n = 0.5, 1, 2, C Pd = 0.9 mM), 1000 µL of 0.2 M, pH = 3, acetic acid-sodium acetate buffer solution containing 0.6 mM TMB, 100 µL of 0.03 M solution of H 2 O 2 , and 200 µL of HQ solution of different concentrations were mixed in a 2 mL PE tube, and then the PE tube was placed in a constant temperature mixer for 5 min at 35 • C and 600 rpm. Finally, its absorbance was measured using a UV-vis spectrophotometer. The relationship between the difference in absorbance and concentration was used as the standard curve for detecting HQ. HQ solution at a concentration of 0-10 mM was added to PE tubes. HQ recovery in tap water and seawater was detected using the established standard curve.
An amount of 50 µL of Van-Pd n NPs (n = 0.5, 1, 2, C Pd = 0.9 mM), 1000 µL of 0.2 M, pH = 3, acetic acid-sodium acetate buffer solution containing 0.6 mM TMB, 100 µL of 0.03 M solution of H 2 O 2 , and 200 µL of HQ solution of different concentrations were added to a 2 mL PE tube. The 2 mL PE tube was placed in a constant-temperature mixer at 30 • C and 600 rpm for 5 min. Finally, the absorbance was measured using a UV-vis spectrophotometer. The recovery of the sample was calculated using the spike recovery formula [25]. The selectivity of Van-Pd 2 NPs was investigated by detecting hydroquinone and potentially interfering substances, such as Mg 2+ , alanine (Ala), phenylalanine (Phe), leucine (Leu), glycine (Gly), proline (Pro), glutamic acid (Glu), maltose (Mal), lactose (Lac), and fructose (Fru). The experimental process was the same as above; the concentration of HQ is 1 mM, and the concentration of other interfering substances is 10 mM.

Biocompatibility of Van-Pd n NPs
The biocompatibility of Van-Pd n NPs (n = 0.5, 1, 2) was determined by MTT. First, cells were added to a 96-well plate at a concentration of 5.0 × 10 3 per well and incubated for 24 h. The DMEM medium containing nanozymes was then replaced with the original medium and incubated for 24 h. The medium was replaced with a thiazole blue (MTT) solution and incubated for 4 h. Finally, the MTT solution was replaced with dimethyl sulfoxide (DMSO), and the absorbance of the 96-well plate was determined using a multifunctional microplate reader.

Characterization of Van-Pd n NPs
The synthesis of Van-Pd n NPs was first characterized by UV-vis spectrophotometry. Figure 1A shows the preparation method of Van-Pd n NPs. As shown in Figure 1B, Na 2 PdCl 4 has an absorption peak at 420 nm in the UV-vis spectrum, which is caused by Pd 2+ . However, there is no characteristic absorption peak of Na 2 PdCl 4 at 420 nm in the spectra of Van-Pd 0.5 NPs, Van-Pd 1 NPs, and Van-Pd 2 NPs. When Pd 2+ is reduced to Pd atoms, the absorption peak at 420 nm disappears [26]. Therefore, Pd 2+ is reduced to Pd atoms in the process of synthesizing Van-Pd n NPs. This indicates the successful preparation of Van-Pd n NPs. a 2 mL PE tube. The 2 mL PE tube was placed in a constant-temperature mixer at 30 °C and 600 rpm for 5 min. Finally, the absorbance was measured using a UV-vis spectrophotometer. The recovery of the sample was calculated using the spike recovery formula [25].
The selectivity of Van-Pd2 NPs was investigated by detecting hydroquinone and potentially interfering substances, such as Mg 2+ , alanine (Ala), phenylalanine (Phe), leucine (Leu), glycine (Gly), proline (Pro), glutamic acid (Glu), maltose (Mal), lactose (Lac), and fructose (Fru). The experimental process was the same as above; the concentration of HQ is 1 mM, and the concentration of other interfering substances is 10 mM.

Biocompatibility of Van-Pdn NPs
The biocompatibility of Van-Pdn NPs (n = 0.5, 1, 2) was determined by MTT. First, cells were added to a 96-well plate at a concentration of 5.0 × 10 3 per well and incubated for 24 h. The DMEM medium containing nanozymes was then replaced with the original medium and incubated for 24 h. The medium was replaced with a thiazole blue (MTT) solution and incubated for 4 h. Finally, the MTT solution was replaced with dimethyl sulfoxide (DMSO), and the absorbance of the 96-well plate was determined using a multifunctional microplate reader.

Characterization of Van-Pdn NPs
The synthesis of Van-Pdn NPs was first characterized by UV-vis spectrophotometry. Figure 1A shows the preparation method of Van-Pdn NPs. As shown in Figure 1B, Na2PdCl4 has an absorption peak at 420 nm in the UV-vis spectrum, which is caused by Pd 2+ . However, there is no characteristic absorption peak of Na2PdCl4 at 420 nm in the spectra of Van-Pd0.5 NPs, Van-Pd1 NPs, and Van-Pd2 NPs. When Pd 2+ is reduced to Pd atoms, the absorption peak at 420 nm disappears [26]. Therefore, Pd 2+ is reduced to Pd atoms in the process of synthesizing Van-Pdn NPs. This indicates the successful preparation of Van-Pdn NPs.  NPs, and Van-Pd2 NPs have good dispersion and a small particle size. Van-Pd0.5 NPs have a particle size of 2.6 ± 0.5 nm, Van-Pd1 NPs have a particle size of 2.9 ± 0.6 nm, and Van-Pd2 NPs have a particle size of 4.3 ± 0.5 nm.  Figure 2 shows TEM images of Van-Pd n NPs (n = 0.5, 1, 2). Van-Pd 0.5 NPs, Van-Pd 1 NPs, and Van-Pd 2 NPs have good dispersion and a small particle size. Van-Pd 0.5 NPs have a particle size of 2.6 ± 0.5 nm, Van-Pd 1 NPs have a particle size of 2.9 ± 0.6 nm, and Van-Pd 2 NPs have a particle size of 4.3 ± 0.5 nm.
DLS is a commonly used way to characterize the hydrodynamic size and zeta potential of nanoparticles in water [27]. Since most of the reaction processes, such as the catalysis of nanozymes, are carried out in aqueous solutions, DLS testing of Van-Pd n NPs (n = 0.5, 1, 2) is required. As can be seen in Figure 3A, the hydrodynamic sizes of nanoparticles of Van-Pd 0.5 NPs, Van-Pd 1 NPs, and Van-Pd 2 NPs were 24.1 nm, 26.1 nm, and 23.6 nm, respectively. The hydrodynamic size of Van-Pd n NPs (n = 0.5, 1, 2) with different molar ratios did not have an obvious difference. Moreover, the zeta potential of the Van-Pd n NPs was tested. As can be seen from Figure 3B, the zeta potentials of Van-Pd 0.5 NPs, Van-Pd 1 NPs, and Van-Pd 2 NPs were −31.5 mV, −30.2 mV, and −32.4 mV, respectively. Among the Van-Pd n NPs (n = 0.5, 1, 2), Van-Pd 2 NPs had the largest absolute value of zeta potential in aqueous solution. DLS is a commonly used way to characterize the hydrodynamic size and zeta potential of nanoparticles in water [27]. Since most of the reaction processes, such as the catalysis of nanozymes, are carried out in aqueous solutions, DLS testing of Van-Pdn NPs (n = 0.5, 1, 2) is required. As can be seen in Figure 3A, the hydrodynamic sizes of nanoparticles of Van-Pd0.5 NPs, Van-Pd1 NPs, and Van-Pd2 NPs were 24.1 nm, 26.1 nm, and 23.6 nm, respectively. The hydrodynamic size of Van-Pdn NPs (n = 0.5, 1, 2) with different molar ratios did not have an obvious difference. Moreover, the zeta potential of the Van-Pdn NPs was tested. As can be seen from Figure 3B, the zeta potentials of Van-Pd0.5 NPs, Van-Pd1 NPs, and Van-Pd2 NPs were −31.5 mV, −30.2 mV, and −32.4 mV, respectively. Among the Van-Pdn NPs (n = 0.5, 1, 2), Van-Pd2 NPs had the largest absolute value of zeta potential in aqueous solution. We selected Van-Pd2 NPs for subsequent XRD to analyze their composition and structure. The diffraction spectra of Figure 4 show that the 2θ values of Van-Pd2 NPs are 39.71°, 46.36°, 67.66°, and 81.42°, respectively. These diffraction angles corresponded to the (111), (200), (220), and (311) faces of Pd, respectively. Compared with the reference code 46-1043 of Pd, it can be seen that the diffraction angle is consistent with the diffraction angle in the reference code, so it can be proved that the nanozyme we synthesized We selected Van-Pd 2 NPs for subsequent XRD to analyze their composition and structure. The diffraction spectra of Figure 4 show that the 2θ values of Van-Pd 2 NPs are  220), and (311) faces of Pd, respectively. Compared with the reference code 46-1043 of Pd, it can be seen that the diffraction angle is consistent with the diffraction angle in the reference code, so it can be proved that the nanozyme we synthesized contained the element Pd. Therefore, we have successfully synthesized Van-Pd 2 NPs through the characterization of XRD. We selected Van-Pd2 NPs for subsequent XRD to analyze their composition and structure. The diffraction spectra of Figure 4 show that the 2θ values of Van-Pd2 NPs are 39.71°, 46.36°, 67.66°, and 81.42°, respectively. These diffraction angles corresponded to the (111), (200), (220), and (311) faces of Pd, respectively. Compared with the reference code 46-1043 of Pd, it can be seen that the diffraction angle is consistent with the diffraction angle in the reference code, so it can be proved that the nanozyme we synthesized contained the element Pd. Therefore, we have successfully synthesized Van-Pd2 NPs through the characterization of XRD.

Characterization of Peroxidase-like Activity
As a commonly used substrate for enzyme activity assays, TMB reacts rapidly to produce blue oxTMB in the presence of reactive oxygen species and a catalyst [28]. Then, UVvis spectrophotometry was used to test the absorbance of the characteristic absorption peak of oxTMB and determine whether it retained a certain enzyme-like activity by comparing the absorbance.
To test whether Van-Pd2 NPs have oxidase-like activity and peroxidase-like activity, we designed the following control groups: TMB + H2O2, Van-Pd2 NPs + H2O2, TMB + Van-Pd2 NPs, and TMB + Van-Pd2 NPs + H2O2. It can be seen from Figure 5A that after 5 min of reaction, the TMB + Van-Pd2 NPs group and the TMB+ Van-Pd2 NPs + H2O2 group had a characteristic absorption peak of oxTMB at 652 nm. The TMB+ Van-Pd2 NPs + H2O2 group showed an excellent characteristic absorption peak at 652 nm; the absorbance was 0.60. The TMB + Van-Pd2 NPs group showed a characteristic absorption peak at 652 nm, but the absorbance was only 0.28. The characteristic absorption peak intensity at 652 nm of the TMB + Van-Pd2 NPs + H2O2 group was 2.14 times that of the only Van-Pd2 NPs group. This indicated that Van-Pd2 NPs had good peroxidase-like activity. In addition,

Characterization of Peroxidase-like Activity
As a commonly used substrate for enzyme activity assays, TMB reacts rapidly to produce blue oxTMB in the presence of reactive oxygen species and a catalyst [28]. Then, UV-vis spectrophotometry was used to test the absorbance of the characteristic absorption peak of oxTMB and determine whether it retained a certain enzyme-like activity by comparing the absorbance.
To test whether Van-Pd 2 NPs have oxidase-like activity and peroxidase-like activity, we designed the following control groups: TMB + H 2 O 2 , Van-Pd 2 NPs + H 2 O 2 , TMB + Van-Pd 2 NPs, and TMB + Van-Pd 2 NPs + H 2 O 2 . It can be seen from Figure 5A that after 5 min of reaction, the TMB + Van-Pd 2 NPs group and the TMB+ Van-Pd 2 NPs + H 2 O 2 group had a characteristic absorption peak of oxTMB at 652 nm. The TMB+ Van-Pd 2 NPs + H 2 O 2 group showed an excellent characteristic absorption peak at 652 nm; the absorbance was 0.60. The TMB + Van-Pd 2 NPs group showed a characteristic absorption peak at 652 nm, but the absorbance was only 0.28. The characteristic absorption peak intensity at 652 nm of the TMB + Van-Pd 2 NPs + H 2 O 2 group was 2.14 times that of the only Van-Pd 2 NPs group. This indicated that Van-Pd 2 NPs had good peroxidase-like activity. In addition, TMB was oxidized to form oxTMB in the TMB + Van-Pd 2 NPs group, indicating the oxidase-like activity of Van-Pd 2 NPs.  Figure 5B, it can be seen that when the amount of vancomycin species is the same, the greater the amount of Pd species, the better the peroxidase-like activity. Compared with the concentration of Pd species at 652 nm, the absorbance of Van-Pd0.5 NPs, Van-Pd1 NPs, and Van-Pd2 NPs reacted with TMB + H2O2 was 0.07, 1.04, and 1.10 after 5 min of reaction, respectively. It can be seen that the absorbance of the Van-Pd2 NPs + TMB + H2O2 group was the highest. This may be because at the same concentration of metal species, the less template used, the fewer active sites were covered, which was more conducive to the redox reaction. Therefore, Van-Pd2 NPs with the best peroxidase-like activity can be used for subsequent experiments. The peroxidase-like activity of Van-Pd2 NPs is affected by a number of external conditions, the main influencing factors of which are pH and temperature. Therefore, we need to investigate the peroxidase-like activity of nanozymes. As shown in Figure 6A, it can be seen that the peroxidase-like activity of Van-Pd2 NPs is set at 100% at pH = 4, and the peroxidase-like activity decreases significantly at other pHs. In addition, Figure 6B is an exploration of the optimal temperature for the peroxidase-like activity of Van-Pd2 NPs. From Figure 5B, it can be seen that when the amount of vancomycin species is the same, the greater the amount of Pd species, the better the peroxidase-like activity. Compared with the concentration of Pd species at 652 nm, the absorbance of Van-Pd 0.5 NPs, Van-Pd 1 NPs, and Van-Pd 2 NPs reacted with TMB + H 2 O 2 was 0.07, 1.04, and 1.10 after 5 min of reaction, respectively. It can be seen that the absorbance of the Van-Pd 2 NPs + TMB + H 2 O 2 group was the highest. This may be because at the same concentration of metal species, the less template used, the fewer active sites were covered, which was more conducive to the redox reaction. Therefore, Van-Pd 2 NPs with the best peroxidase-like activity can be used for subsequent experiments.
The peroxidase-like activity of Van-Pd 2 NPs is affected by a number of external conditions, the main influencing factors of which are pH and temperature. Therefore, we need to investigate the peroxidase-like activity of nanozymes. As shown in Figure 6A, it can be seen that the peroxidase-like activity of Van-Pd 2 NPs is set at 100% at pH = 4, and the peroxidase-like activity decreases significantly at other pHs. In addition, Figure 6B is an exploration of the optimal temperature for the peroxidase-like activity of Van-Pd 2 NPs. Van-Pd 2 NPs have the best peroxidase-like activity at 35 • C. Therefore, we can determine that the optimal conditions for Van-Pd 2 NPs are pH 4 and a temperature of 35 • C. The peroxidase-like activity of Van-Pd2 NPs is affected by a number of external conditions, the main influencing factors of which are pH and temperature. Therefore, we need to investigate the peroxidase-like activity of nanozymes. As shown in Figure 6A, it can be seen that the peroxidase-like activity of Van-Pd2 NPs is set at 100% at pH = 4, and the peroxidase-like activity decreases significantly at other pHs. In addition, Figure 6B is an exploration of the optimal temperature for the peroxidase-like activity of Van-Pd2 NPs. Van-Pd2 NPs have the best peroxidase-like activity at 35 °C. Therefore, we can determine that the optimal conditions for Van-Pd2 NPs are pH 4 and a temperature of 35 °C.

Characterization of Van-Pd2 NPs Catalytic Kinetics
In order to explore the catalytic activity of peroxidase-like Van-Pd2 NPs, it is required to study their catalytic reaction kinetic characterization. The reaction kinetics of

Characterization of Van-Pd 2 NPs Catalytic Kinetics
In order to explore the catalytic activity of peroxidase-like Van-Pd 2 NPs, it is required to study their catalytic reaction kinetic characterization. The reaction kinetics of nanozymes are determined by changing the concentrations of the substrates TMB and H 2 O 2 . The test data were analyzed using the Lineweaver-Burk equation to obtain the data in Figure 7. Moreover, the peroxidase-like activity of other materials was compared with Van-Pd 2 NPs shown in Table 1. The Michael's constant K m and the maximum reaction rate V max are then calculated according to the equation [34]. Firstly, the concentration of substrate TMB was changed within 0.04-0.4 mM, and the K m value and V max value of Van-Pd 2 NPs were 1.007 mM and 13.6 × 10 −8 Ms −1 , respectively. Then, the concentration of H 2 O 2 was changed within 0.2-2.0 mM, and the K m value and V max value of Van-Pd 2 NPs were 0.623 mM and nanozymes are determined by changing the concentrations of the substrates TMB and H2O2. The test data were analyzed using the Lineweaver-Burk equation to obtain the data in Figure 7. Moreover, the peroxidase-like activity of other materials was compared with Van-Pd2 NPs shown in Table 1.  The Michael's constant Km and the maximum reaction rate Vmax are then calculated according to the equation [34]. Firstly, the concentration of substrate TMB was changed within 0.04-0.4 mM, and the Km value and Vmax value of Van-Pd2 NPs were 1.007 mM and 13.6 × 10 −8 Ms −1 , respectively. Then, the concentration of H2O2 was changed within 0.2-2.0 mM, and the Km value and Vmax value of Van-Pd2 NPs were 0.623 mM and 12.566 × 10 −8 Ms −1 , respectively. Table 1 is a comparison of the catalytic performance of different nanozymes. Compared with other nanozymes, such as Au-NCs and Pb 2+ for H2O2, the Km value of Van-Pd2 NPs for H2O2 is small. This indicates that Van-Pd2 NPs have good H2O2 affinity, which is conducive to the formation of reactive oxygen species and promotes the subsequent catalytic reaction. Thus, Van-Pd2 NPs have good peroxidase-like catalytic kinetics.

Mechanism of Van-Pd 2 NPs Peroxidase-like Activity
To delve into the mechanism of the catalytic process, additional research has been conducted. Figure 8C shows the fluorescence spectrum of the reaction mixture containing terephthalic acid (TA) and Van-Pd 2 NPs after the addition of hydrogen peroxide (H 2 O 2 ). Terephthalic acid is commonly used as a fluorescent probe to detect the presence of hydroxyl radicals (•OH). In the presence of •OH, terephthalic acid undergoes a reaction to form 2-hydroxy terephthalic acid (TAOH), which exhibits strong fluorescence at 435 nm when excited at a 315 nm wavelength. This fluorescence emission at 435 nm indicates the formation of TAOH due to the reaction between •OH and terephthalic acid.
The purpose of using terephthalic acid as a probe in this experiment is to investigate whether •OH radicals are produced by Van-Pd 2 NPs through the catalytic decomposition of hydrogen peroxide. The fluorescence signal at 435 nm confirms the presence of •OH radicals, suggesting that the peroxidase-like activity of Van-Pd 2 NPs is indeed due to the generation of •OH radicals. As depicted in Figure 8A, upon the addition of Van-Pd 2 NPs into the solution containing TA and H 2 O 2 , it was observed that the fluorescence intensity curve of TAOH exhibited a notably higher value compared to the other groups. In Figure 8B, The peak value obtained from the group TA + H 2 O 2 + Van-Pd 2 NPs was measured at 304, which was approximately double the peak value of the TA + H 2 O 2 group without Van-Pd 2 NPs. These experimental findings strongly indicate that the catalytic mechanism of Van-Pd 2 NPs primarily involves the generation of •OH.
conducted. Figure 8C shows the fluorescence spectrum of the reaction mixture containing terephthalic acid (TA) and Van-Pd2 NPs after the addition of hydrogen peroxide (H2O2). Terephthalic acid is commonly used as a fluorescent probe to detect the presence of hydroxyl radicals (•OH). In the presence of •OH, terephthalic acid undergoes a reaction to form 2-hydroxy terephthalic acid (TAOH), which exhibits strong fluorescence at 435 nm when excited at a 315 nm wavelength. This fluorescence emission at 435 nm indicates the formation of TAOH due to the reaction between •OH and terephthalic acid. The purpose of using terephthalic acid as a probe in this experiment is to investigate whether •OH radicals are produced by Van-Pd2 NPs through the catalytic decomposition of hydrogen peroxide. The fluorescence signal at 435 nm confirms the presence of •OH radicals, suggesting that the peroxidase-like activity of Van-Pd2 NPs is indeed due to the generation of •OH radicals. As depicted in Figure 8A, upon the addition of Van-Pd2 NPs into the solution containing TA and H2O2, it was observed that the fluorescence intensity curve of TAOH exhibited a notably higher value compared to the other groups. In Figure  8B, The peak value obtained from the group TA + H2O2 + Van-Pd2 NPs was measured at 304, which was approximately double the peak value of the TA + H2O2 group without Van-Pd2 NPs. These experimental findings strongly indicate that the catalytic mechanism of Van-Pd2 NPs primarily involves the generation of •OH.

HQ Detection
HQ is a reducible biomass that reduces oxTMB to TMB. Therefore, we can use the excellent peroxidase-like activity of Van-Pd2 NPs to establish the standard curve for HQ detection. This experimental system is Van-Pd2 NPs + TMB + H2O2 + HQ, and then UV-vis

HQ Detection
HQ is a reducible biomass that reduces oxTMB to TMB. Therefore, we can use the excellent peroxidase-like activity of Van-Pd 2 NPs to establish the standard curve for HQ detection. This experimental system is Van-Pd 2 NPs + TMB + H 2 O 2 + HQ, and then UV-vis spectrophotometry is used to detect the UV-vis absorption spectrum of the solution, as shown in Figure 9A. The absorbance at 652 nm was measured by UV-vis spectrophotometry. As shown in Figure 9B, the absorbance at 652 nm gradually increases in a linear manner with the increasing concentration of HQ. However, when the concentration of HQ solution is greater than 1 mM, the absorbance at 652 nm tends to be stable. Figure 9C shows that the standard equation for HQ detection was Y = 0.0313 + 2.6812 × C HQ (R 2 = 0.9973). As shown in Table 2, the linear range was 0.001-0.1 mM, and the detection limits were 0.323 µM. Compared to the reported GCN-Cu NFs [36], the detection range is 0.82-100 µM, and the detection limit is 0.82 µM. Van-Pd 2 NPs have high sensitivity and a wide detection range. Mg 2+ , alanine, phenylalanine, leucine, glycine, proline, glutamic acid, maltose, lactose, and fructose were used to test the selectivity of this assay. The other components caused very weak absorbance changes, as depicted in Figure 9D. These findings indicate that the method employed in the study exhibited high selectivity for the detection of glutathione.
Polymers 2023, 15,3148 10 of 14 spectrophotometry is used to detect the UV-vis absorption spectrum of the solution, as shown in Figure 9A.   In addition, Van-Pd 2 NPs can be used to determine the recovery of samples. The sample is first added to different solutions to make a spiked solution. A certain amount of spiked solution was added to the reaction solution of Van-Pd 2 NPs + TMB + H 2 O 2 . The absorbance of the solution was determined using UV-vis spectrophotometry. The spike recovery formula was then used to determine the recovery of the sample. From the comparison of the data in Table 3, it can be seen that the detection and recovery of HQ in seawater and river water are 108% and 98%, respectively. Therefore, the detection of HQ by Van-Pd 2 NPs had high accuracy.

Biocompatibility
Biological vancomycin was used in the preparation of Van-Pd 2 NPs. In addition to testing the nanozyme activity and catalytic performance of Van-Pd 2 NPs, it is also necessary to determine the biocompatibility of Van-Pd 2 NPs. The MTT method is a commonly used method for determining the biocompatibility of samples. Van-Pd 2 NPs with A549 cells were incubated for 24 h. Then, the cell viability of Van-Pd 2 NPs was determined by MTT to determine the biocompatibility of nanozymes. The results are shown in Figure 10, and we set the Van-Pd 2 NPs group, the vancomycin group, and the blank group. The cell viability in the vancomycin group remained at 90%, and the cell viability in the Van-Pd 2 NPs group reached 85% at 200 µg/mL. By comparison with the cell activity of the blank group, Van-Pd 2 NPs and vancomycin have almost no cytotoxicity. Therefore, it is known that Van-Pd 2 NPs have good biocompatibility through cell viability assays.

Conclusions
In summary, we successfully synthesized Van-Pd2 NPs with good peroxidase-like activity by the biological template method. The catalytic kinetics of Van-Pd2 NPs conformed to the typical Michaelis-Menten equation, and there is a good affinity for H2O2 in peroxidase-like activity. The prepared nanozyme Van-Pd2 NPs were used to establish a simple and reliable detection method for HQ. The detection range was determined to be 1-100 μM, with a detection limit of 0.323 μM. Van-Pd2 NPs and vancomycin were noncytotoxic. Therefore, the colorimetric detection method with high selectivity has a good application prospect in the detection of HQ.

Conclusions
In summary, we successfully synthesized Van-Pd 2 NPs with good peroxidase-like activity by the biological template method. The catalytic kinetics of Van-Pd 2 NPs conformed to the typical Michaelis-Menten equation, and there is a good affinity for H 2 O 2 in peroxidase-like activity. The prepared nanozyme Van-Pd 2 NPs were used to establish a simple and reliable detection method for HQ. The detection range was determined to be 1-100 µM, with a detection limit of 0.323 µM. Van-Pd 2 NPs and vancomycin were non-cytotoxic. Therefore, the colorimetric detection method with high selectivity has a good application prospect in the detection of HQ.